A murine model of dengue virus infection in suckling C57BL/6 and BALB/c mice

Abstract

Dengue is a significant public health concern across tropical and subtropical regions worldwide, principally causing disease in children. Very young children are at increased risk of severe manifestations of dengue infection. The mechanism of dengue disease in this population is not fully understood. In this study, we present a murine model of dengue virus primary infection in suckling C57BL/6 and BALB/c mice in order to investigate disease pathogenesis. Three-day-old C57BL/6 mice intraperitoneally infected with DENV-2 NGC were more susceptible to infection than BALB/c mice, showing increased liver enzymes, extended viremia, dissemination to organs and histological alterations in liver and small intestine. Furthermore, the immune response in DENV-infected C57BL/6 mice exhibited a marked Th1 bias compared to BALB/c mice. These findings highlight the possibility of establishing an immunocompetent mouse model of DENV-2 infection in suckling mice that reproduces certain signs of disease observed in humans and that could be used to further study age-related mechanisms of dengue pathogenesis.

Keywords: BALB/c; C57BL/6; dengue virus; mouse model; suckling mice.

FIGURE 1

Timeline of DENV infection and determinations in suckling mice. C57BL/6 and BALB/c pups were infected IP with 5 × 10⁵ PFU/g body weight of DENV‐2 NGC or C6/36 cell culture supernatant (control) at 3 PND. Letters indicate the hours post inoculation (HPI) at which mice were euthanized for the following determinations: A, Plasma levels of aspartate aminotransferase, alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatine kinase (CK), and platelet counts; B, Total RNA was extracted from plasma, mesenteric lymph nodes, liver, kidney, spleen, small intestine, and brain and viral RNA was quantified using qRT‐PCR; C, Liver, kidney, spleen, small intestine, and brain were fixed, embedded in paraffin and stained with hematoxylin and eosin (H&E); D, Splenocytes were incubated during 72 h with DENV‐2 antigen or medium, and after centrifugation, IFN‐γ and IL‐4 were quantified in cell culture supernatants. In addition, total RNA in cell pellets was used to determine mRNA expression of T‐bet and GATA3. In order to reach minimum volumes of plasma for some determinations, blood from 2 mice was pooled

FIGURE 2

Intraperitoneal inoculation of suckling mice. Three‐day‐old C57BL/6 and BALB/c mice were inoculated with 5 × 10⁵ PFU/g of DENV‐2 or cell supernatant by an inverse IP administration technique. A, Mouse immobilization and location of inoculation site. B, Inoculum incision in the subcutaneous level moving forward into cranium‐caudal direction and entering the peritoneal cavity for inoculum administration

FIGURE 3

Effect of DENV‐2 infection on suckling mouse growth and survival. Growth of C57BL/6 (A) and BALB/c (B) infected and control mice until 8 d PI. Survival of C57BL/6 (C) and BALB/c (D) infected and control mice until 8 d PI. Values represent means ± SEM. *P < .05, determined by Student's unpaired t test

FIGURE 4

Biochemical parameters at 8 d PI in suckling mice infected with DENV‐2. Plasma levels of AST (A, B), ALT (C, D), LDH (E, F) and CK (G, H) were determined in C57BL/6 and BALB/c infected and control mice. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; LDH, lactate dehydrogenase. Values represent means ± SEM. *P < .05, determined by Student's unpaired t test

FIGURE 5

Virus detection by qRT‐PCR in plasma and tissues after DENV‐2 infection in suckling mice. C57BL/6 and BALB/c mice were infected with DENV‐2 IP, and at different times PI (0, 17, 24, 48, 72, 96, 120 and 168 h), mice were euthanized and plasma (A, B), mesenteric lymph nodes (C) and organs (D, E) were collected. Total RNA was extracted from tissues and DENV RNA was detected by qRT‐PCR. Abbreviations: LN, lymph node. Values represent means ± SEM. *P < .05, **P < .01 determined by one‐way ANOVA (dashed line) and Bonferroni's multiple comparison post‐test (solid line). Dotted lines show limits of detection

FIGURE 6

Histopathology in suckling mice after DENV‐2 infection. Cross sections of various tissues of infected and control C57BL/6 and BALB/c mice at 8 d PI. Arrows indicate lymphoid mononuclear infiltrates in liver and small intestine and lytic focuses in the apical region of intestinal villi in DENV‐2 infected mice. Magnification: 200× for liver and kidney, and 100× for spleen, small intestine and brain. H&E staining

FIGURE 7

Immune response in suckling mice after DENV‐2 infection. C57BL/6 and BALB/c mice were infected IP with DENV‐2 or C6/36 cell supernatant (control). At 8 d PI, splenocytes were isolated from infected and control mice and stimulated with UV‐DENV‐2 during 72 h. A, C, Cytokine levels were measured in cell supernatants by immunoassay. B, D, Total RNA was extracted from cell pellets and relative quantity (QR) of transcription factor mRNA corresponding to Th1 and Th2 cell pathways was determined. Values represent means ± SEM. *P < .05, determined by one‐way ANOVA (dashed line) and Bonferroni's multiple comparison post‐test (solid line). Dotted lines show limits of detection